Antiphospholipid antibody binding to bilayer-coated glass microspheres

Abstract

Thrombosis, recurrent fetal loss, and thrombocytopenia are clinical manifestations associated with circulating antibodies that recognize cardiolipin (CL)- or phosphatidylserine (PS)-dependent antigens. Enzyme-linked immunosorbent assays (ELISAs) are generally used to determine the presence and specificity of antiphospholipid antibodies (aPLs). However, the presentation of the phospholipid antigen in the ELISA assay is unknown. In this study, we determined the specificity of three mouse monoclonal aPLs for phospholipid bilayer membranes. These monoclonal aPLs had been characterized by ELISA to have different specificities for CL and PS and were designated BA3B5C4 ( CL + PS + ), 3SB9b ( CL − PS + ), and D11A4 ( CL + PS − ). Bilayers composed of 0–100% PS or CL in phosphatidylcholine (PC) were formed on the surface of 1.6 μm diameter glass microspheres to permit analysis by flow cytometry. BA3B5C4 and 3SB9b bound specifically to both PS- and CL-containing bilayers, and binding increased with increasing percentage of anionic phospholipid. The threshold for PS-dependent binding was 20 mol% PS for both BA3B5C4 and 3SB9b. For CL-dependent binding, the threshold was below 25 mol% CL for both of these antibodies. Binding to PS-containing bilayers was tested as a function of ionic strength for BA3B5C4 and 3SB9b. The ionic strength dependence of the binding suggested that the intermolecular attractive forces between anti-PS antibodies and PS-containing bilayers are predominantly multiple weak electrostatic bonds. D11A4 bound only to bilayers composed of 100% PS and 100% PC, and this antibody did not bind to CL-containing bilayers. The binding specificities of these aPLs to bilayer membranes suggest that, in this system, the conformation of the epitope involving CL, and perhaps PS, is different from that expressed in the routine clinical ELISA. Two of the monoclonal antibodies reacted in this model system at the low levels of PS typically externalized in the plasma membranes of activated platelets, apoptopic lymphocytes, and senescent red blood cells: thus, these surfaces are plausible candidates for the site of pathologically relevant antibody interactions.